Defining Microbiota-Derived Metabolite Butyrate as a Senomorphic: Therapeutic Potential in the Age-Related T Cell Senescence

Abstract

Advancing age is accompanied by an accumulation of senescent T cells that secrete pro-inflammatory senescence-associated secretory phenotype (SASP) molecules. Gut-microbiota-derived signals are increasingly recognised as immunomodulators. In the current study, we demonstrated that ageing and the accumulation of senescent T cells are accompanied by a reduction in microbial-derived short-chain fatty acids (SCFAs). Culturing aged T cells in the presence of butyrate suppresses the induction of a senescence phenotype and inhibits the secretion of pro-inflammatory SASP factors, such as IL6 and IL8. Administration of faecal supernatants from young mice rich in butyrate prevented in vivo accumulation of senescent spleen cells in aged mice. The molecular pathways governing butyrate's senomorphic potential include a reduced expression of DNA damage markers, lower mitochondrial ROS accumulation, and downregulation of mTOR activation, which negatively regulates the transcription factor NFκB. Our findings establish butyrate as a potent senomorphic agent and provide the evidence base for future microbiome restitution intervention trials using butyrate supplements for combating T cell senescence, ultimately reducing inflammation and combating age-related pathologies to extend lifelong health.

Keywords: T cell; ageing; cellular senescence; inflammation.

FIGURE 1

Stool butyrate levels correlate with peripheral accumulation of senescent T cells in older adults and characterisation of Senomorphic properties of butyrate and attenuation of SASP phenotype Box whisper plots showing (A) serum (B) stool butyrate levels in healthy young individuals (blue; n = 36) and healthy aged participants (n = 39) as measured by high‐performance liquid chromatography (C) Scatterplots show the correlation between stool butyrate levels and senescent CD8 T cell frequency in the peripheral blood of healthy young individuals (blue; n = 36) and healthy aged participants (n = 39). (D) Schematic of experimental design indicating culturing of aged T cells in CD3 coated plates with SCFA butyrate for 72 h (n = 6) and collection of cell‐culture supernatants for determining SASP features by ELISA and assessment of features of senescence via flow cytometry (E) CD3 treated T cells were incubated in the absence of butyrate (control), positive control (1 mM sodium bicarbonate) and with different concentrations of butyrate (0.5–2 mM) for 72 h. At the end of treatment, IL6 concentration in ell‐culture supernatant was measured by ELISA (n = 5) and (F) cell death was assessed via Annexin V positive staining. The secretion of (G) IL6 (H) IL8 (I) IL1β from CD3 treated aged T cells in the presence of 1 M butyrate. (J) IL6^+ve senescent CD57^+ve CD4 T cells CD3 treated aged T cells in the presence of 1 M butyrate. Data are mean ± SEM of six independent experiments. Statistical analysis was performed by two tailed paired student's t‐test *p < 0.05, **p < 0.01.

FIGURE 2

Regulation of senescence phenotype by butyrate in aged CD8 T cells (A) Representative flow cytometry showing gating strategy for T cell subsets based on phenotypic expression of CD45RA/CCR7 staining. In vitro culture of PBMCs in CD3‐coated wells for 3 days in the presence/absence of SCFA butyrate. On Day 3 (B) frequency of phosphorylated p53 expressing CD8 T cell subsets (C) phospho p53 expression levels in CD8 T cell subsets (D) frequency of γH2AX expressing CD8 T cell subsets (E) γH2AX expression levels (MFI) in CD8 T cell subsets. Statistical analysis was performed using a two‐tailed paired Student's t‐test. (F) Representative image stream images of cells stained with cell surface marker CD8, nuclear DAPI stain, and DNA damage marker γH2AX and merged image when PBMCs were cultured in CD3‐coated wells for 3 days in the presence/absence of SCFA butyrate. (G) frequency of SA‐βGal expressing T cell. The bar charts show expression data as the mean ± SEM of six experiments. *p = 0.05.

FIGURE 3

Effect of butyrate on nuclear factor kappa B (NFκB), p‐38 MAPK, mTOR signalling, and autophagic flux in aged CD8 T cells. In vitro culture of T cells in CD3‐coated wells for 3 days in the presence/absence of SCFA butyrate for (A) frequency of phosphorylated NF‐kB expressing CD8 T cell subsets, (B) NF‐kB expression levels in CD8 T cell subsets, (C) frequency of phosphorylated p38 expressing CD4 T cell subsets, (D) p‐p38 expression levels in CD4 T cell subsets, (E) pS6 mean fluorescence intensity (MFI), (F) autophagic flux in CD8 T cell subsets (naïve, central memory, effector memory, EMRA). A two‐tailed paired Student's t‐test performed the statistical analysis. Data are shown as the mean ± SD *p = 0.05, **p < 0.001.

FIGURE 4

Effect of butyrate on mitochondrial mass and ROS production in CD8 T cells. In vitro culture of T cells in CD3‐coated wells for 3 days in the presence/absence of SCFA butyrate and stimulation. (A) MitoTracker green expression levels in aged CD8 T cell subsets (B) Representative histogram of the shift in MitoTracker green MFI between control (blue) and butyrate (red). (C) Representative ImageStream images of cells stained with cell surface marker CD3 and mitochondrial mass indicator MitoTracker green and subsequent merged images. (D) MitoSOX red expression levels in aged CD8 T cell subsets. Statistical analysis was performed by a two‐tailed paired Student's t‐test. Data are shown as the mean ± SD *p < 0.05, **p < 0.01.

FIGURE 5

Transcriptome signature of aged T cells post butyrate treatment Heat map showing expression levels of differentially expressed genes in T cells isolated from six healthy aged participants cultured in CD3‐coated plates in the absence (control) or with 1 mM butyrate for 72 h.

FIGURE 6

Influence of transfer of a young stool faecal filtrate to aged mice on the accumulation of senescent cells in the spleen (A) Overview of the experimental plan (B) Immunohistochemistry staining of the spleen with DAPI (nuclear stain) demonstrating white pulp regions (brighter blue regions) × 4 magnification (C) CD3 expressing T cell zone (red) × 10 magnification (D–F) merged images with arrows showing senescence marker p53 labelled with FITC (green) in CD3 expressing T cell zone × 40 magnification. (G) Graph representing the number of p53‐positive cells in the T cell zone of the spleen in 8 weeks young C57BL/6J mice (n = 4), aged C57BL/6J mice (n = 4) and aged mice receiving faecal filtrate from young mice = 4. Values are expressed as mean ± SEM. (H) RT‐q PCR‐mediated gene expression analysis of p53 (H) and p 16 (I) pro‐inflammatory cytokine IL6 (J) in the spleen of mice (n = 4 per group). *p = 0.05, **p < 0.001.